Most recombinant DNA work to date has been carried out with Escherichia coli (E. coli). E. coli is a member of the gram-negative class of bacteria which contain two layers of membranes enclosing a periplasmic space. Many of the products produced in E. coli are secreted into this periplasmic space, if secreted at all. Few products are secreted outside the living cells into the growth medium.
On the other hand, Bacillus subtilis (B. subtilis) is a member of the gram-positive class of bacteria which contain only a single layer of bacterial membrane. Thus B. subtilis can produce large amounts of protein which are secreted directly into the growth medium. Moreover, production of proteins in B. subtilis is advantageous since the organism is non-pathogenic and does not produce endotoxins. In addition, B. subtilis has been extensively studied and is the archetype for genetic studies among gram-positive microorganisms.
Although the general approach to gene cloning in E. coli is applicable to B. subtilis, attempts to produce a useful product of a heterologous gene cloned into B. subtilis and secreted into the growth medium have been retarded and made especially difficult because of the general lack of suitable cloning and expression vectors.
This paucity of expression vectors is explained in part by the lack of recognition of foreign transcription and translation initiation signals in B. subtilis. Consequently, the well known trp (Hallewell, R.A. and S. Emtage, Gene 9, 24-47 [1980]), lac (K. ItaKura et al., Science 198, 1056-1063 [1977]; Roberts, T.M. et al., Proc. Nat. Acad. Sci. USA 76, 5596-5600 [1979], lpp (Lee, N. et al., J. Bacteriol. 146, 861-866 [1981]; Zwiebel, L.J. et al., J. Bacteriol. 145, 654-656 [1981]and Natamura, K. and M. Inouye, Cell 18, 1109 [1979]) and bacteriophage .lambda. P.sub.L (Bernard, H. et al., Gene 5, 59-76 [1979]) transcription and translation-directing systems are not functional in B. subtilis.
Thus, with the exception of a few drug resistance genes from gram-positive organisms such as staphylococcus and streptococcus, few foreign genes encoding prokaryotic and eukaryotic proteins have been expressed in Bacillus, especially B. subtilis, (for review see "Genetics and Biotechnology of Bacilli", eds. A. T. Ganesan and J. A. Hock; Academic Press, Inc. [1984]and dissertation of J. Palva,, infra). Moreover, the expression yield is in general small, and therefore the development of superior expression vectors having potent promoters for Bacillus subtilis has been desired.
At present, the known Bacillus subtilis promoters with the respective base sequences clarified include the veg promoter, tms promoter, pen P promoter (C.P. Moran Jr. et al., Mol. Gen. Genetics 186, 339-346 [1982]), spo VC promoter (C.P. Moran Jr. et al., Nucl. Acids Res. 9, 5979-5990 [1981]), spo VG promoter (C.P. Moran Jr. et al., Cell 25, 783-791 [1981]), .phi. 29 G3a promoter, O 29 G3b promoter, O 29 G2 promoter, O 29 A1 promoter (C.L. Murray and J.C. Rabinowitz, J. Biol. Chem. 257, 1053-1062 [1982]), pMG 102 promoter, pMG 201 promoter (M.Z. Gilman et al., Nucl. Acids Res. 9, 5991-6000 [1981]), spo 1-15 promoter (G. Lee et al., J. Mol. Biol. 139, 407-422 [1980]), spo 1-16 promoter (G. Lee et al., Molec. Gen. Genetics 180, 57-65 [1980]), and SPO2 promoter (R.G. Schoner et al., Gene 22, 47-57 [1983]). Among them, the SPO2 promoter (R.G. Schoner et al., supra) and the veg promoter (European patent application, publication no. 116411) are the only promoters that have actually been utilized in gene expression.